Carefully regulated antioxidant defense mechanisms are critical to the survival of all aerobic organisms. Reductions in antioxidant defenses result in oxidative injury to components of the cell and, if extensive, to cell death. Muscle as a tissue is particularly vulnerable to oxidative injury because of its high metabolic rate (and thus high rate of production of reactive species) and its high content of iron-containing proteins. There is considerable evidence that oxidative stress contributes to the degenerative changes that occur in muscle disease, in muscle injury from exercise or ischemia, in muscle wasting syndromes, and in age-related muscle atrophy. The focus of the studies of this proposal is the regulation of antioxidant defenses in muscle and how changes in that regulation may contribute to muscle cell injury in muscular dystrophies. Specific Aim 1 focuses on the regulation of muscle antioxidant gene expression in response to oxidative stress, addressing particularly the role of the redox-sensitive transcriptional regulators NFKB and AP- 1. These studies will all be done in vitro and will involve a combination of gel shift analysis, reporter gene assays, and mutational analysis of the 5' flanking region of genes encoding antioxidant enzymes. Specific Aim 2 addresses the mechanism of injury in muscle in which there is dysregulation of antioxidant gene expression. We propose a model to explain the biphasic nature of the effects of overexpression of Cu,Zn-superoxide dismutase (protective at low levels of expression, destructive at high levels of expression). We will assess steady-state levels of reactive oxygen species in cells expressing different levels of the enzyme, and we will use genetic techniques both in vitro and in vivo to investigate the biphasic response. The studies of Specific Aim 3 explore the mechanism of increased susceptibility to oxidative stress of muscles that have abnormalities in dystrophin expression which, in vivo, cause muscular dystrophies. We will test the hypothesis that the increased susceptibility of these cells to oxidative stress is due to altered regulation of cellular glutathione, a critical mediator of cellular antioxidant defenses. We will also use microarray ("DNA chip") technology to examine differences in gene expression between normal and dystrophin-deficient muscle, focusing exclusively on the pre-necrotic phase of the disease in the mdx (dystrophin-deficient) mouse. Using this approach, we will test our specific hypothesis of the role of oxidative injury in the pathogenetic process and we will determine if the results of the expression array analysis suggest alternate hynotheses.